Biopharmaceutical aggregation assessment and counterfeit detection using magnetic resonance relaxometry

ABSTRACT

The present invention generally relates to a method of using NMR relaxation rates (R 2 ) of water molecules as an indicator of the extent of aggregation of biopharmaceutical formulations. The biopharmaceutical can be evaluated nondestructively without the vial or container being opened or protective seal compromised (i.e., broken). The method is applicable to all biopharmaceuticals and the water signal obtained by magnetic resonance relaxometry is very strong and sensitive because water is used as the solvent and is present in high (&gt;90%) concentrations in every biopharmaceutical formulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 16/682,157 filed on Nov. 13, 2019, now U.S.Pat. No. 10,724,974, which is a divisional of U.S. patent applicationSer. No. 16/369,534 filed on Mar. 29, 2019, now U.S. Pat. No.10,514,347, which is a divisional of U.S. patent application Ser. No.14/780,711 filed on Sep. 28, 2015, now U.S. Pat. No. 10,267,754, whichwas filed under the provisions of 35 U.S.C. § 371 and claims priority ofInternational Patent Application No. PCT/US2014/033833 filed on Apr. 11,2014, which in turn claims priority to U.S. Provisional PatentApplication No. 61/811,401 filed Apr. 12, 2013 in the name of YihuaBruce Yu entitled “Assessing Biopharmaceutical Aggregation UsingMagnetic Resonance Relaxometry,” all of which are incorporated byreference herein in their entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant NumberU01FD004320 awarded by the U.S. Food and Drug Administration and GrantNumber CBET1133908 awarded by the National Science Foundation. Thegovernment has certain rights in the invention.

FIELD

The present invention relates to methods for assessing the extent ofbiopharmaceutical aggregation and to methods of detecting counterfeitbiopharmaceuticals using magnetic resonance relaxometry.

DESCRIPTION OF THE RELATED ART

Aggregation is a significant problem in biopharmaceutical manufacturingand storage. Aggregated proteins have lower or no biological activity,are more likely to elicit an immune response, may have an alteredbiodistribution, and may have altered pharmacokinetics.Disadvantageously, most protein or peptide aggregation is not visible tothe naked eye and can only be detected by scientific instruments.

Problems of quality control of pharmaceutical products in aqueoussolutions become more and more pressing with the fast growing number ofdrugs based on biomacromolecules (e.g., proteins, nucleic acids,polysaccharides, etc.). One of the earliest signs of degradation ofaqueous pharmaceutical preparations is the aggregation of the activefactor during the manufacturing or long-term storage of the product [1].Even in the past, when the number of protein-based biotherapeutics wasquite limited (e.g., to insulin, γ-globulin, albumin, etc.), themonitoring of aggregation was a time-consuming and complex process [2].In the modern pharmaceutical industry, the main techniques to detectsuch soluble aggregates include size-exclusion chromatography (SEC),analytical ultracentrifugation sedimentation velocity (AUC-SV), dynamiclight scattering (DLS), electrophoresis, and field-flow fractionation[2]. Disadvantageously, all of these conventional techniques could beregarded as “destructive,” since their application by definitionrequires the opening of the vial and renders the drug unusable even ifit is not considered degraded. Moreover, these conventionally usedtechniques are time-consuming, which precludes their efficientapplication for high-throughput quality checks post-production.

Recent breakthrough developments in the instrumentation for nuclearmagnetic resonance (NMR) spectroscopy and imaging have opened upopportunities to design novel nondestructive analytical techniques forthe pharmaceutical industry. Using NMR relaxation measurements,high-throughput NMR screening has been actively used to identifymolecules with a high affinity to a receptor at very low concentrations[3]. The analytical procedures become significantly faster with theapplication of commercially available computer-controlled NMRautosamplers. Of special importance was the appearance of commerciallyavailable, relatively inexpensive benchtop NMR and magnetic resonanceimaging (MM) instruments and relaxometers [4]. These instruments,working at the magnetic field strength of 0.3-0.5 T have alreadydemonstrated their extreme versatility in the chemical and foodindustry, and very recently, in the monitoring of drug delivery steps invitro, for example, the hydration and swelling of controlled releasetablets [4]. Benchtop NMR relaxometers enable highly accuratemeasurements of nuclear spin relaxation times T₁ and T₂. Moreover, mostof these instruments have a permanent or electronically cooled magnetwith the variable bore from 10 mm to 25 mm which provides a greatflexibility in the measurements of vials of various sizes.

Clearly, there is a need for a fast and reliable technique which wouldallow one to nondestructively measure the extent of degradation of thepharmaceutical preparation and still retain its usability. Towards thatend, the present invention relates to a method of using NMR relaxationrates (R₁ and R₂) of water molecules as an indicator of the aggregationof biopharmaceutical formulations. Advantageously, the method describedherein is not drug specific, is easy to use, is nondestructive, andfast. Further, the NMR relaxation rates (R₁ and R₂) of water can be usedto detect counterfeit biopharmaceutical-containing compositions by usingthe magnetic resonance relaxometry of water to obtain field strengthdependency of the relaxation rate (relaxation dispersion profile) todetect the deviations in the content of ingredients in the complexmixtures of biopharmaceuticals in order to identify whether thebiopharmaceuticals are counterfeited.

SUMMARY

The present invention generally relates to a method of using NMRrelaxation rates (R₁ and R₂) of water molecules as an indicator of theaggregation of biopharmaceutical formulations. The biopharmaceutical canbe evaluated without the vial or container being opened or protectiveseal compromised (i.e., broken). The method is applicable to allbiopharmaceuticals and the signal obtained by magnetic resonancerelaxometry is very strong and sensitive because water is used as thesolvent in high (>90%) concentrations in every biopharmaceuticalformulation.

The present invention also relates to a method of using NMR relaxationrates (R₁ and R₂) dispersion profile of water molecules to detectcounterfeit compositions, for example, counterfeitbiopharmaceutical-containing compositions.

In one aspect, a method of measuring the extent of aggregation of abiopharmaceutical in a biopharmaceutical-containing product isdescribed, said method comprising measuring the transverse relaxationrate of water R₂ in the biopharmaceutical-containing product, andextrapolating the extent of aggregation of the biopharmaceutical from astandard calibration curve using the measured R₂ of water.

In another aspect, a method of detecting counterfeit compositionscomprises: measuring a relaxation dispersion profile R(ω) of thecomposition, and comparing the measured R(ω) to a standard R(ω) for thatcomposition, wherein the composition is a counterfeit if the measuredR(ω) is greater than an indicated percent difference from the standardR(ω), wherein the indicated percent difference is provided by themanufacturer of the composition, and wherein the measurement of therelaxation dispersion profile R(ω) is done non-destructively. Thecomposition can be a biopharmaceutical-containing product.

Other aspects, features and advantages of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) illustrates SEC chromatograms of BSA solution (75 μM) at 25°C. prior to heating.

FIG. 1(b) illustrates SEC chromatograms of BSA solution (75 μM) at 25°C. after 15 min at 60° C.

FIG. 1(c) illustrates SEC chromatograms of BSA solution (75 μM) at 25°C. after 30 min at 60° C.

FIG. 1(d) illustrates the peak fit analysis of the expanded SECchromatogram from FIG. 1(b) using Origin 8.1.

FIG. 2 illustrates the inability to visibly see aggregates formed uponheating with the naked eye.

FIG. 3(a) illustrates the pulse sequence for a saturation-recoveryexperiment.

FIG. 3(b) illustrates the pulse sequence for a CPMG experiment. Theflip-angle of the excitation pulse (small-angle) varies depending on thestrength of the water signal.

FIG. 4(a) illustrates the dependence of the longitudinal relaxation rateof water R₁ on the concentration of BSA, and the buildup of theaggregates after the solutions with different initial concentrationswere exposed to heat (60° C.).

FIG. 4(b) illustrates the dependence of the transverse relaxation rateof water R₂ on the concentration of BSA, and the buildup of theaggregates after the solutions with different initial concentrationswere exposed to heat (60° C.).

FIG. 5(a) illustrates the dependence of the longitudinal relaxation rateconstant of water R₁ on the concentration of BSA aggregates at 60° C.

FIG. 5(b) illustrates the dependence of the transverse relaxation rateof water R₂ on the concentration of BSA aggregates at 60° C. Thenumbered labels correspond to vials 2, 3 and 4 shown in FIG. 2.

FIG. 6(a) illustrates the transverse relaxation rate of water R₂relative to the percent aggregation of BSA.

FIG. 6(b) illustrates the transverse relaxation rate of water R₂relative to the mean molecular weight in kDa of BSA aggregates.

FIG. 7(a) illustrates the transverse relaxation rate of water R₂relative to the percent aggregation of γ-globulin.

FIG. 7(b) illustrates the transverse relaxation rate of water R₂relative to the mean molecular weight in kDa of γ-globulin aggregates.

FIG. 8 illustrates the transverse relaxation rate of water R₂ relativeto the variation of the percentage of HPC and γ-globulin in thecomposition.

DETAILED DESCRIPTION, AND PREFERRED EMBODIMENTS THEREOF

The present invention generally relates to a method of using NMRrelaxation rates (longitudinal and transverse relaxation rate constants,R₁ and R₂, respectively) of water molecules as an indicator of theextent of aggregation of biopharmaceuticals inbiopharmaceutical-containing products. Further, the present inventionrelates to a method of detecting counterfeit compositions, e.g.,biopharmaceutical-containing compositions, using the NMR relaxationrates (R₁ and R₂) of water.

Advantageously, the method described herein is a reliable and simplemethod to detect biopharmaceutical aggregation and hence, viability, andhas application as a quality control tool for biopharmaceuticals. Themethod enables the calculation of the extent of aggregation inbiopharmaceuticals non-destructively, without opening the vial orstorage container, by measuring the nuclear spin relaxation rateconstant, R₁ and R₂, of water as a quality control parameter. Thenuclear spin relaxation rate constant can be measured before dispensingthe biopharmaceutical to the patient to confirm that the extent ofaggregation is within an acceptable range. Accordingly, this methodredefines the conventional methods of evaluating shelf-life ofbiopharmaceuticals by assessing their precise extent of aggregation foreach vial or storage container.

As defined herein, the “biopharmaceutical” includes antibodies,proteins, peptides, nucleic acids, polysaccharides, and combinationsthereof.

As used herein, “counterfeit” compositions, e.g., biopharmaceuticals,correspond to compositions sold or provided under a specific brand orgeneric name that are not manufactured by or on behalf of the owner ofthe brand name or a provider of a generic composition. Often thecounterfeit compositions are purchased online at a lower cost and arenot made according to the exact manufacturing standards of the owner ofthe brand name or provider of a generic composition and/or are lessefficacious than the brand name or generic composition.

As defined herein, a “vial” corresponds to a small glass (optionally,plastic, ceramic, etc., anything nonmetal) vessel, bottle or ampouleused to store the biopharmaceutical. The vial can have a screw top, atop that is closed using a cork or plastic stopper, a crimp vial (closedwith a rubber stopper and a metal cap), a flip-top or snap cap. The vialcan be tubular, or have a bottle-like shape with a neck. Other types andshapes of vials used to store biopharmaceuticals as well as cappingmeans are readily understood by the person skilled in the art.

As defined herein, a “non-destructive” measurement is defined as ameasurement performed without opening the vial or otherwise accessingthe contents of the vial (for example by withdrawing a portion through arubber gasket). Moreover, a non-destructive measurement means that noadditives or probes or the like are added to the vial prior to themeasurement of the transverse relaxation rate of water R₂ in thebiopharmaceutical-containing product.

The present inventors have surprisingly discovered that the relaxationrate (R₁ and R₂) of water increases depending on the extent ofbiopharmaceutical aggregation and the size distribution of theaggregates. Because water is present in every biopharmaceuticalformulation, this method provides a universally applicable method toassess the degree of biopharmaceutical aggregation using magneticresonance relaxometry. The NMR/MRI-based nondestructive techniqueinvolves the measurement of the NMR relaxation rates of water moleculesin biopharmaceutical products. Such measurements can be performed usingcommercially available, relatively inexpensive benchtop NMR and MRIinstruments and relaxometers and are “nondestructive” and“non-disruptive” in that they could be performed without opening thevial and as such, after the analysis, the biopharmaceuticals ofacceptable quality could still be used. That said, it should beappreciated that the measurements can occur destructively as well,whereby the vial is opened.

Accordingly, in one aspect, a method of measuring the extent ofaggregation of a biopharmaceutical in a biopharmaceutical-containingproduct, said method comprising measuring the transverse relaxation rateof water R₂ in the biopharmaceutical-containing product, andextrapolating the extent of aggregation of the biopharmaceutical from astandard calibration curve using the measured R₂ of water. The extent ofaggregation can be the percent aggregation of the biopharmaceutical, theconcentration of the biopharmaceutical aggregates, or the molecularweight of the biopharmaceutical aggregates.

The transverse relaxation rate of water can be used as a quality controlparameter for biopharmaceuticals, wherein the water relaxivity ismeasured during manufacturing at a specified temperature and the resultlisted in the package insert of the biopharmaceutical product.Distributors, hospitals and pharmacies can then use benchtop NMR or MRIinstruments and relaxometers to measure water relaxivity at a specifiedtemperature and compare it with the value listed in the package insertbefore distribution or usage. If the extent of aggregation is outside ofan acceptable limit, the biopharmaceutical product should not bedistributed or used.

Further, the method described herein is fast and reliable and allows theuser to nondestructively measure the extent of aggregation, and hencedegradation, of the pharmaceutical preparation and still retain itsusability if no aggregation is detected. The method is not drug specificand is easy to use.

A non-exclusive list of biopharmaceutical products that can be measuredfor the extent of aggregation includes, but is not limited to, bovineserum albumin; human serum albumin; human γ-globulin; hormones such asinsulin, glucagon, gonadotrophins, and growth hormone; haematopoieticgrowth factors such as erythropoetin; blood factors such as Factor VIIIand Factor IX; thrombolytic agents; interferons such as interferon-α,interferon-β, and interferon-γ; interleukin-based products such asinterleukin-2; vaccines such as the influenza vaccine; monoclonalantibodies such as adalimumab, rituximab, infliximab, trastuzumab,ustekinumab, denosumab, and golimumab, and including fragments ofmonoclonal antibodies (e.g., Fc and Fab fragments), variants ofmonoclonal antibodies, such as single-chain antibodies, bivalentantibodies, and the like, and polyclonal antibody preparations forresearch or clinical use including therapeutic antibody preparationssuch as intravenous immunoglobulin (IVIG); tumor necrosis factor;abatacept; alefacept; etanercept; denileukin diftitox; OPTISON;NEUPOGEN; albumin; and ribonuclease A, to name a few.

In practice, the standard for aggregation in the biopharmaceuticalsample should be determined by the manufacturer. The manufacturer canprovide the calibration curve (line) of relaxation rate R₂ versus theextent of aggregation (e.g., percent aggregation) measured at a giventemperature (e.g., 25° C.). It could be either in the form of a formulaor the graphic (like nomogram, etc.). The user will then measure the R₂of water of the biopharmaceutical sample at the same temperature andcompare the value with the standard calibration curve, as understood bythe person skilled in the art. The manufacturer will also provide amaximum acceptable extent of aggregation value or maximum water R₂ valuefor the biopharmaceutical, relative to the extent of aggregation at thetime of production, whereby the biopharmaceutical is overly degraded(i.e., no longer viable) and should not be dispensed. It should beappreciated that the standard calibration curve can be prepared by theuser, if necessary, and that the maximum acceptable extent ofaggregation will vary according to the biopharmaceutical.

It should be appreciated by the person skilled in the art that themethods described herein can be used to measure the extent ofaggregation of a biopharmaceutical in a biopharmaceutical-containingproduct during pre-formulation, formulation, production, orpost-production.

In another aspect, a method of determining if a biopharmaceutical in abiopharmaceutical-containing product remains viable is described, saidmethod comprising measuring the transverse relaxation rate of water R₂in the biopharmaceutical-containing product, extrapolating the extent ofaggregation of the biopharmaceutical from a standard calibration curveusing the measured R₂ of water, and comparing the extent of aggregationto the maximum acceptable extent of aggregation for thebiopharmaceutical, relative to the extent of aggregation at the time ofproduction, to determine viability of the biopharmaceutical. The extentof aggregation can be the percent aggregation of the biopharmaceutical,the concentration of the biopharmaceutical aggregates, or the molecularweight of the biopharmaceutical aggregates.

In another aspect, a kit for determining the extent of aggregation of abiopharmaceutical in a biopharmaceutical-containing product isdescribed, said kit comprising instructions on measuring the transverserelaxation rate of water R₂ in a biopharmaceutical-containing product, astandard calibration curve for the biopharmaceutical, and a maximumacceptable extent of aggregation, wherein the maximum acceptable extentof aggregation for the biopharmaceutical is determined relative to theextent of aggregation of the biopharmaceutical at the time ofproduction.

In yet another aspect, a method of preparing the standard calibrationcurve for a biopharmaceutical is described, said method comprising:

selecting a temperature whereby the biopharmaceutical will experience ahigher rate of aggregation;

exposing the biopharmaceutical to the temperature for at least two,three, four, or five time periods;

measuring the transverse relaxation rate of water R₂ in thebiopharmaceutical at the at least two, three, four, or five timeperiods;

measuring the percent aggregation of the biopharmaceutical using sizeexclusion chromatography at the at least two, three, four, or five timeperiods; and plotting the percent aggregation relative to the transverserelaxation rate of water R₂ and determining the best-fit regressionline.

With regards to this aspect of the invention, for the purposes ofstandard calibration curve, the concentration of the aggregates and/orthe percentage of aggregation are measured by means of size-exclusionchromatography, it would be appreciated by those skilled in the art thatother methods could be also employed including, but not limited to,ultracentrifugation, dynamic light scattering, small-angle X-ray and/orneutron scattering. Also, the person skilled in the art will easilyrecognize other techniques to induce aggregation of thebiopharmaceuticals in addition to heat-induced aggregation, such aslight-induced, ultrasound-induced, and shaking, to name a few. All abovetechniques are incorporated herein in their entirety by reference.

In still another aspect, the NMR relaxation rates (R₁ and R₂) of watercould be used to detect counterfeit compounds, e.g., biopharmaceuticalsand/or counterfeit complex mixtures of said bioparmaceuticals, in acomposition, e.g., a biopharmaceutical-containing product. It iswell-known to the person skilled in the art that NMR relaxation rates(R₁ and R₂) are field-dependent, i.e., they both depend on magneticfield strength and hence, the resonance frequency ω [5]. Suchdependency, in the form of R(ω) is called a relaxation dispersionprofile. The technique to measure R(ω) is called magnetic resonancerelaxometry (MRR). The inventors surprisingly discovered that thetransverse relaxation rate constant of water R₂ is very sensitive towarda variety of conditions of a complex mixture, in particular, thecomposition of a mixture of biopharmaceuticals, drugs, cosmetics, etc.In this aspect, the MRR of water signal will be used to detect thedeviations in the content of ingredients in the complex mixtures inorder to identify whether the mixtures are counterfeited. In practice,the unique composition will be characterized by the unique relaxationdispersion profile R(ω), a “magnetic fingerprint” measured by MRR andprovided by the manufacturer of the proprietary composition, optionally,placed on the manufacturer's website. The user then could measure therelaxation dispersion profile R(ω) of a composition purchased on themarket under the manufacturer's brand name and after comparing it withthe “magnetic fingerprint” of said composition would be able to identifypotentially counterfeited compositions. Such measurements arenondestructive in that they could be performed without opening the vialand accordingly, after the analysis the compositions which are notcounterfeited could still be used. Measurements of the relaxationdispersion profile R(ω) could be performed using relatively inexpensivebenchtop relaxometers for MRR experiments, such as, for example,SMARtracer FFC relaxometer operated in the range from 2.5 mT to 250 mT(STELAR s.r.l, Mede, Italy). Accordingly, the method of detectingcounterfeit compositions comprises: measuring a relaxation dispersionprofile R(ω) of the composition, and comparing the measured R(ω) to astandard R(ω) for that composition, wherein the composition is acounterfeit if the measured R(ω) is greater than an indicated percentdifference from the standard R(ω), wherein the indicated percentdifference is provided by the manufacturer of the composition, andwherein the measurement of the relaxation dispersion profile R(ω) isdone non-destructively. For example, the method can be used fordetecting counterfeit biopharmaceutical-containing products, whereinsaid method comprises: measuring a relaxation dispersion profile R(ω) ofthe biopharmaceutical-containing product, and comparing the measuredR(ω) to a standard R(ω) for that biopharmaceutical-containing product,wherein the biopharmaceutical-containing product is a counterfeit if themeasured R(ω) is greater than an indicated percent different from thestandard R(ω), wherein the indicated percent difference is provided bythe manufacturer, and wherein the measurement of the relaxationdispersion profile R(ω) is done non-destructively. It should beunderstood by the person skilled in the art that the indicated percentdifference calculated as an average deviation between the measured andstandard R(ω) dispersion profile may be a percentage such as 0.1%, 0.5%,1%, 2%, 3%, 4%, or 5%, depending on the composition, e.g.,biopharmaceutical-containing product, and known variations that mayoccur during storage and transportation.

The features and advantages of the invention are more fully shown by theillustrative examples discussed below.

EXAMPLE 1

Solutions of bovine serum albumin (BSA) in phosphate buffered saline(PBS) were used as a model system for protein-based pharmaceuticals. BSAis readily commercially available and is also widely used in manymedical applications [6]. The NMR relaxivities of water (R₁ and R₂) weremeasured in freshly prepared protein solutions and in the solutions witha controlled degree of protein aggregation. To mimic protein aggregationin biopharmaceutical products, a heat-induced aggregation method wasused [7]. The slow aggregation process in model systems that occursduring prolonged standing at room temperature was also evaluated.

Analytical grade reagents for buffer preparation, solvents and bovineserum albumin (BSA, lyophilized, 98% purity, molecular weight 66 kDa)were purchased from Sigma Aldrich Inc., St. Louis, Mo., and were usedwithout further purification. All solutions were prepared in phosphatebuffered saline (PBS) containing 50 mM sodium phosphate and 100 mMsodium chloride in ultrapure H₂O (resistance 18 MOhm) at pH 7.4.

Heat-induced aggregation of BSA in aqueous solution was monitored usingSEC. To study the formation of BSA aggregates at different time pointsof exposure to heat, the solutions of BSA in PBS buffer at eightdifferent concentrations (30 μM, 75 μM, 110 μM, 150 μM, 230 μM, 310 μM,380 μM, 455 μM) were heated in water baths at 60° C. for 15 min and 30min, respectively. After cooling down to 25° C., the concentration ofthe BSA aggregates was measured by size-exclusion chromatography usingthe Äkta FPLC system (Amersham Pharmacia Biotech, Inc., Uppsala, Sweden)with UPC-900 UV-detector set at 280 nm. Column: Superose 12 100/300 GL(GE Healthcare, Little Chalfont, UK), 24 mL bed volume, particle size 1μm, 3 kDa molecular weight cutoff. Injection sample volume: 200 μL.Eluent: PBS buffer at pH 7.4 with 1% sodium azide. Flow rate: 0.5mL/min. Concentrations of the initial pure (non-aggregated) BSA and itsaggregates after the exposure to heat were estimated from thechromatographic peaks areas using the subroutine Peak Fit Analysis inOrigin 8.1 (OriginLab Co., Northhampton, Mass.). Quality of thenonlinear peak fit was always R²=0.97-0.99.

FIG. 1 illustrates the SEC chromatograms of BSA at 30 μM before andafter heating, wherein FIG. 1(a) shows the aggregation at 25° C. priorto heating, FIG. 1(b) shows the aggregation after 15 min at 60° C., andFIG. 1(c) shows the aggregation after 30 min at 60° C. It can be seenthat the peaks that correspond to larger aggregates grow at the expenseof the peak to non-aggregated BSA (designated by the asterisk).Deconvolution of the SEC chromatogram of FIG. 1(b) makes it possible toanalyze the extent of aggregation. To calculate the concentration of theBSA aggregates, formula (1) is used:

$\begin{matrix}{\lbrack C\rbrack_{agg} = {\frac{S_{tot} - S_{BSA}}{S_{tot}}\lbrack C\rbrack}_{BSA}} & (1)\end{matrix}$where S_(tot) is the sum of the areas of all peaks resulting from themultiple peak analysis (see, solid line in FIG. 3(d)), S_(BSA) is thearea of the peak from non-aggregated BSA (the peak labeled with theasterisk), [C]_(BSA) is the initial concentration of BSA prior toheating, and [C]_(agg) is the concentration of BSA aggregates. Thegoodness of fitting parameter is R²=0.99. The concentrations of the BSAaggregates, as determined using formula (1) after 15 min and 30 min at60° C., are tabulated in Table 1.

TABLE 1 Concentrations of the BSA aggregates after 15 min and 30 min ofthe heat-induced aggregation at 60° C. Initial BSA BSA aggregates after15 BSA aggregates after 30 concentration, μM min at 60° C., μM min at60° C., μM 30 13 15 75 39 45 106 65 69 151 95 98 227 150 162 303 176 184379 234 242 455 255 263

In order to study the effect of protein aggregation on the longitudinalR₁ and transverse R₂ relaxation rate constants of water, diluted(10⁻⁵-10⁻⁴ M) solutions of bovine serum albumin (BSA) were used as amodel system for protein-based preparations. Albumins are known to formaggregates under short (minutes) exposure to moderate temperatures(50-70° C.) [8]. The aggregates formed upon heating are not visible tothe naked eyes (see, FIG. 2, wherein vial 0 is PBS; vial 1 is 450 μM BSAin PBS, never heated; vial 2 is 230 μM BSA in PBS, heated for 30 min at60° C.; vial 3 is 150 μM BSA in PBS, heated for 30 min at 60° C.; andvial 4 is 75 μM BSA in PBS, heated for 30 min at 60° C.).

To study how the relaxation rate constants of water, R₁ and R₂,correlate with the extent of protein aggregation, BSA solutions of eightdifferent concentrations (30 μM, 75 μM, 110 μM, 150 μM, 230 μM, 310 μM,380 μM, 450 μM, same as in SEC measurements) were prepared. Eachsolution was divided into three portions: one portion stayed at 25° C.;one portion was heated to 60° C. for 15 min and then cooled back to 25°C. for measurement; one portion was heated to 60° C. for 30 min and thencooled back to 25° C. for measurement. All NMR experiments were carriedon a 400 MHz (9.4 T) Varian NMR spectrometer.

Because water is the solvent, its NMR signal is very strong. To avoidthe radiation damping effect in relaxation measurements of water,saturation-recovery and CPMG sequence with a small flip-angle excitationpulse were applied [8]. The flip-angle of the excitation pulse(small-angle) varies depending on the strength of the water signal.

To measure the longitudinal relaxation rate constant R₁,saturation-recovery experiments were used. Referring to FIG. 3(a), therelaxation delay (d1) was 15 s. A 2 second pre-saturation pulse wasapplied to saturate the water signal before the recovery time. Tendifferent T₁ recovery times (d2=0.1, 0.2, 0.35, 0.6, 1, 1.8, 3, 5, 8 and12 s) were applied to get a fully recovered water signal. Thelongitudinal relaxation time T₁ (=1/R₁) can be extracted by fitting theexperimental data to Formula (2):I(t)=I ₀×[1−exp(−t/T ₁)]  (2)where I(t) is the ¹H₂O signal intensity at time t, I₀ is the ¹H₂O signalintensity when the signal is fully recovered, and t is the T₁ recoverytime.

To measure the transverse relaxation rate constant R₂,Carr-Purcell-Meiboom-Gill (CPMG) experiments were used. Referring toFIG. 3(b), the relaxation delay (d1) is 15 s, the interval between 180°pulses is 120 μs (2τ=120 μs). A small-angle excitation pulse (flip angleabout)10° was used to prevent the strong water signal from overflowingthe receiver. The said flip-angle of the excitation pulse (small-angle)varies depending on the strength of the water signal. Ten different T₂delay times (2nτ, n is the number of 180°-pulses) was used to getsufficient signal decay (about 90%). The CPMG delay ‘2nτ’ was optimizedbased on the T₂ value of each sample (to get a properly distribution of10 data point in the whole signal decay curve). For example, for thesample with a BSA concentration of 455 μM, the 10 CPMG delay times wereset to: 0.1, 0.25, 0.4, 0.6, 0.8, 1.0, 1.3, 1.6 ,2.0 ,2.4 s. For thesample with a BSA concentration of 30 μM, the 10 CPMG delays were setto: 0.1, 0.3, 0.5, 0.8, 1.1, 1.5, 1.9, 2.5, 3.0, 3.7, 4.5 and 5.5second. The transverse relaxation time T₂ (=1/R₂) value can be extractedby fitting experimental data to Formula (3):I(t)=I ₀×exp(−t/T ₂)  (3)where I(t) is the ¹H₂O signal intensity at time t, I₀ is the initial¹H₂O signal intensity when t=0, and t is the T₂ delay time.

The measurements of R₁ of water in the aggregated BSA solutionsdemonstrated virtually no dependence on the exposure time to heat andthe concentration of the aggregates (FIG. 4(a)). Contrary to R₁, thetransverse relaxation rate constant R₂ of water has grown markedly afterheat exposure, and grows even more with the heat exposure time, andhence, with the increase in the concentration of BSA aggregates (see,FIG. 4(b)). As seen from FIG. 4(b), at higher initial concentrations ofBSA (300-400 μM), the exposure to heat leads to significant, almosttwo-fold increase in the water relaxation rate constant R₂.

FIGS. 5(a) and 5(b) illustrate the relationship between [C]_(agg) andwater R₁ and R₂, respectively. It can be seen that R₁ is ratherinsensitive toward aggregation. In contrast, R₂ increases linearly with[C]_(agg). Further, this linear dependency shows indifference to theduration of heating, suggesting that R₂ depends only on the extent ofaggregation, not on how the aggregation was achieved.

The above results conclusively demonstrate the possibility of using thetransverse relaxation rate constant R₂ of water to detect proteinaggregation. As water is used as the solvent in all biopharmaceuticalpreparations, this technique can be used to quantify biopharmaceuticalaggregation non-destructively. Such detections can be accomplished usingNMR spectrometers, MM scanners, or bench-top MR relaxometers (e.g.,BT-NMR/BT-MRI) [4]. The advantage of this approach is that it does notrequire opening the vial and hence is entirely non-destructive andnon-disruptive. Because water is present in every biopharmaceuticalpreparations at very high concentration, this method is universallyapplicable to biopharmaceuticals and is amenable to full automationusing bench-top relaxometers.

EXAMPLE 2

The initial concentration of all proteins in Example 2 for the study ofheat-induced aggregation was 15 mg/mL which for BSA corresponds to 0.2mM, and for γ-globulin corresponds to 0.1 mM. As in the Example 1, allsolutions were prepared in PBS buffer at pH 7.4. Bovine serum albumin(BSA, lyophilized, 98% purity, molecular weight 66 kDa) and γ-globulin(from human blood, >99% purity, molecular weight 150 kDa) were purchasedfrom Sigma Aldrich Inc., St. Louis, Mo., and were used without furtherpurification. The temperatures used to induce aggregation were 55° C.(square, in FIGS. 6 and 7) and 60° C. (circle, in FIGS. 6 and 7) andexposure times at these temperatures varied from 2 minutes to 30 minutes(2, 5, 10, 15, 20, 25, and 30 minutes at each temperature). Differentexposure times were used to attain different degrees of aggregation. Thepercentage of the aggregation was determined by SEC using the BioLogicDuoFlow® FPLC (Bio-Rad Laboratories, Hercules, Calif.) with UV-detectorset at 280 nm. Column: Bio-Sil SEC (Bio-Rad Laboratories, Hercules,Calif.), 14 mL bed volume, particle size 5 μm, 5 kDa molecular weightcutoff. Injection sample volume: 100 μL. Eluent: PBS buffer at pH 7.4with 1% sodium azide. Flow rate: 0.7 mL/min. Concentrations of theinitial pure (non-aggregated) BSA and γ-globulin and their aggregatesafter the exposure to heat were estimated from the chromatographic peaksareas using the subroutine Peak Fit Analysis in Origin 8.1 (OriginLabCo., Northhampton, Mass.). Quality of the nonlinear peak fit was alwaysR²=0.97-0.99. The mean molecular weight of the aggregates was determinedfrom dynamic light scattering (DLS) data based on the observed sizedistribution of the detected particles. The light-scattering experimentswere carried out with light-scattering setup provided by PhotocorInstruments (College Park, Md.) with He-Ne laser, photomultiplier andautomatic goniometer. The sample in a cell was submerged into thesilicon oil to ensure the uniform temperature of the measurements (25°C.) and to reduce possible stray light. Scattering profiles weredetected at scattering angle θ=90°. The resulting auto-correlationfunction was processed using DynaLS software (SoftScientific, TiratCarmel, Israel) to obtain size distributions of the aggregates and theirmean molecular weights.

The results for BSA are shown in FIGS. 6(a) and 6(b) and the results forγ-globulin are shown in FIGS. 7(a) and 7(b). The best-fit regressionline based on the data at the respective temperatures with a high R²value (0.99) can be seen in all four figures, which verifies that if theuser has a formula including the slope of the best-fit regression lineor the figure itself and makes a non-destructive measurement of R₂ ofwater in the biopharmaceutical-containing product, they can determinethe extent of aggregation and determine if the biopharmaceutical isoverly degraded and unusable.

EXAMPLE 3

As introduced hereinabove, the present inventors surprisingly discoveredthat the transverse relaxation rate constant of water R₂ is verysensitive toward a variety of conditions of a complex mixture. Forexample, compositions comprising 10 mg/mL BSA with varying amounts ofγ-globulin and hydroxypropylcellulose (HPC) were prepared, wherein thepercentage of BSA (10 mg/mL) was held constant at 33% while the combinedpercentage of γ-globulin and HPC are held constant at 67%. As in theExamples 1 and 2, all solutions were prepared in PBS buffer at pH 7.4.Bovine serum albumin (BSA, lyophilized, 98% purity, molecular weight 66kDa) and γ-globulin (from human blood, >99% purity, molecular weight 150kDa) were purchased from Sigma Aldrich Inc., St. Louis, Mo., and wereused without further purification. The measurement of the transverserelaxation rate of water R₂ was carried out as described hereinabove at25° C.

The results are shown in FIG. 8, where it can be seen that thetransverse relaxation rate of water R₂ varies with changes in the makeupof the complex mixture. Knowing this, changes in the makeup of allcomplex compositions should result in variations in the transverserelaxation rate of water R₂ and hence permit the identification ofcounterfeit compositions.

EXAMPLE 4

The present inventors also determined that the transverse relaxationrate of water R₂ is sensitive to the chemical modifications of peptides.For example, both N-terminal acetylation and C-terminal amidationinduced readily detectable change in water R₂ (see, e.g., Table 2). Notethat N-terminal acetylation, which blocks a positive charge, wasimplemented in a peptide with 6 positively changed amino acids whileC-terminal amidation, which blocks a negative charge, was implemented ina peptide with 6 negatively charged amino acids. Hence, eachmodification turned off 1 out of 7 charges. The fact that the transverserelaxation rate of water R₂ varied, attests to the sensitivity of thismethod.

TABLE 2 Impact of peptide N-, C-terminal blockage on water R₂ (8 mMpeptide, pH 7.4) N-terminal acetylation charge water R₂ (s⁻¹) C-terminalamidation charge water R₂ (s⁻¹) H₂N—KW(KA)₃KWK- +7 1.15acetyl-EW(EA)₃EWE- −7 0.53 amide OH acetyl-KW(KA)₃KWK- +6 1.33acetyl-EW(EA)₃EWE- −6 0.60 amide amide Charge refers to the total numberof chargeable groups in a peptide (side chains + terminals)

The results are shown in Table 2, where it can be seen that thetransverse relaxation rate of water R₂ varies with chemicalmodifications Knowing this, minor modifications to compounds incompositions should result in variations in the transverse relaxationrate of water R₂ and hence permit the identification of counterfeitcompositions.

Although the invention has been variously disclosed herein withreference to illustrative embodiments and features, it will beappreciated that the embodiments and features described hereinabove arenot intended to limit the invention, and that other variations,modifications and other embodiments will suggest themselves to those ofordinary skill in the art, based on the disclosure herein. The inventiontherefore is to be broadly construed, as encompassing all suchvariations, modifications and alternative embodiments within the spiritand scope of the claims hereafter set forth.

REFERENCES

-   1. Krayukhina, E.; Uchiyama, S.; Nojima, K.; Okada, Y.; Hamaguchi,    I.; Fukui, K., J. Biosci. Bioeng. 2012, 115, 104-110.-   2. Oliva, A.; Fariña, J. B.; Llabrés, M., Int. J. Pharm. 1996, 143,    163-170.-   3. Dalvit, C.; Flocco, M.; Knapp, S.; Mostardini, M.; Perego, R.;    Stockman, B. J.; Veronesi, M.; Varasi, M., J. Am. Chem. Soc. 2002,    124, 7702-7709.-   4. Metz, H.; Mäder, K., Int. J. Pharm. 2008, 364, 170-178.-   5. Bloembergen, N., Purcell, E. M., Pound, R. V., Phys. Rev. 1948,    73, 679-746.-   6. Fanali, G.; di Massi, A.; Trezza, V.; Marino, M.; Fasano, M.;    Ascenzi, P., Mol. Asp. Med. 2012, 33, 209-290.-   7. Saso, L.; Valentini, G.; Grippa, E.; Leone, M. G.; Silvestrini,    B., Res. Commun. Pathol. Pharmacol. 1998, 102, 15-28.-   8. Mao, X.; Ye, C., Concepts Magn. Reson. 1997, 9, 173.

What is claimed is:
 1. A method of assessing the quality of apost-production product comprising a biopharmaceutical, said methodcomprising: measuring a transverse relaxation rate of water R₂ of thepost-production product at a specified temperature; and comparing themeasured R₂ of water of the post-production product to a maximum R₂ ofwater of the product at the specified temperature at the time ofproduction, wherein if the measured R₂ of water of the post-productionproduct is greater than the maximum R₂ of water for the product at thetime of production, the product should not be distributed or used. 2.The method of claim 1, wherein the product is contained in a vial. 3.The method of claim 2, wherein the transverse relaxation rate of waterR₂ is measured without opening the vial or otherwise accessing thecontents of the vial containing the product.
 4. The method of claim 2,wherein the transverse relaxation rate of water R₂ can be measuredwithout adding any additives to the vial containing the product.
 5. Themethod of claim 1, wherein the biopharmaceutical is selected from thegroup consisting of a protein, a peptide, a nucleic acid, and apolysaccharide.
 6. The method of claim 1, wherein the biopharmaceuticalis selected from the group consisting of a hormone, a blood factor, aninterferon, a haematopoietic growth factor, an interleukin-basedproduct, a vaccine, and a monoclonal antibody.
 7. The method of claim 1,wherein the biopharmaceutical is selected from the group consisting ofbovine serum albumin, human serum albumin, human γ-globulin, insulin,glucagon, gonadotrophins, growth hormone, erythropoetin, Factor VIII,Factor IX, thrombolytic agents, interferon-α, interferon-β,interferon-γ, interleukin-2, influenza vaccine, adalimumab, rituximab,infliximab, trastuzumab, ustekinumab, denosumab, golimumab, Fcfragments, Fab fragments, intravenous immunoglobulin (IVIG), tumornecrosis factor, abatacept, alefacept, etanercept, denileukin diftitox,OPTISON, NEUPOGEN, albumin, and ribonuclease A.
 8. The method of claim1, wherein the R₂ is measured using nuclear magnetic resonance (NMR) ormagnetic resonance imaging (MRI).
 9. The method of claim 1, wherein thecontrol composition comprises at least one compound having a knownconcentration and sample composition is represented as comprising thesame compound(s) having the same concentration(s).